Polymers having phosphorylcholine groups have been researched as biocompatible polymers, and biocompatible materials prepared by coating various base agents with such polymers have been developed.
For example, Patent Document 1 discloses a cosmetic in which powder coated with a homopolymer or copolymer of 2-methacryloyloxyethyl phosphorylcholine is used as cosmetic powder to improve moisture retention and adhesion to the skin.
Also, Patent Document 2 and Patent Document 3 disclose medical materials and a separation agent coated with polymers having phosphorylcholine groups.
The surface of the aforementioned materials are coated with a polymer obtained by polymerizing monomers having the phosphorylcholine structure prepared by reacting an acrylic-type monomer mainly having hydroxyl groups with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and then using trimethylamine to turn the reaction product into a quaternary ammonium (refer to Patent Documents 4 and 5 for the preparation method).
Patent Document 4 describes the preparation of a copolymer of 2-methacroyloxyethylphosphorylcholine and methacrylate, and Patent Document 5 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.
On the other hand, there are many commercially available packings for GFC, which separates biological samples such as proteins and polypeptides that have smaller molecular weights than proteins by means of size exclusion. For the packings for GFC, there are packings that use cross-linked hydrophilic polymers as the carrier and packings that use silica gel as the carrier.
A packing that uses a cross-linked hydrophilic polymer as the carrier has a wide pH range for the mobile phase and is highly versatile. However, compared with a packing that uses silica as the carrier, a packing that uses a polymer as the carrier is (1) harder to obtain a higher theoretical plate number due to the difficulty in controlling the fine pore size. Also, a packing that uses a polymer as the carrier is (2) often times incapable of obtaining chromatograms with good reproducibility due to an inferior strength against the high pressure applied when used for high-performance liquid chromatography (HPLC) and the swelling of the particles caused by the mobile phase solvent.
A packing that uses silica gel as the carrier has the problem of adsorption of proteins and/or polypeptides onto the surface of the silica gel carrier. To address this problem, there are packings commercially available that use silica gel whose surface is modified with non-dissociative hydrophilic groups for the purpose of suppressing the adsorption of proteins and/or polypeptides in the analysis sample onto the silica gel.
For example, Shodex PROTEIN KW-803 (product name) is commercially available from Showa Denko KK as a silica gel-type GFC column. This silica gel-type column is described in the catalogue as a silica gel-type GFC mode column suitable for analyzing proteins having a molecular weight of several thousands to a million.
Also, YMC-Pack Diol (product name) is commercially available from YMC Co. Ltd. This is also described as a silica gel-type GFC column prepared by chemically bonding functional groups having the diol structure to a silica gel carrier; it can be used to separate proteins having a molecular weight of ten thousand to several hundred thousand.
Non-Patent Document 1 reports a reduction in protein adsorption due to phosphorylcholine groups chemically grafted onto the carrier.
Patent Documents 6 and 7 disclose an organic silane type surface modifier (silane coupling agent) having the betaine structure, which is known to show excellent hydrophilicity. According to Patent Document 6, a silane coupling agent having sulfobetaine composed of the positive charge of the quaternary ammonium and the negative charge of sulfonic acid can be obtained by reacting dimethylaminoalkyl silane with 1,3-propanesulfone in an organic solvent. Patent Document 7 describes a method of manufacturing a silane coupling agent having carboxybetaine composed of quaternary ammonium and a carboxyl group. These silane coupling agents, when applied and dried on glass and such, can modify the surface of the object. However, although the betaine of such structures can give superior hydrophilicity to the object surface, electric neutrality is not achieved due to the uneven strength of the positive charge and the negative charge in betaine. For example, sulfobetaine is negatively charged due to the strong acidity of sulfonic acid and carboxybetaine shows a positive charge due to quaternary ammonium. The betaine structure of this kind has a very strong ion exchange interaction with protein, which leads to irreversible adsorption of the protein.
There is no example of these silane coupling agents used for chromatography packings and filters and experimental devices for the purpose of biocompatibility and/or protein adsorption suppression.
Patent Document 1: Japanese Patent Laid-Open H7-118123 bulletin
Patent Document 2: Japanese Patent Laid-Open 2000-279512 bulletin
Patent Document 3: Japanese Patent Laid-Open 2002-98676 bulletin
Patent Document 4: Japanese Patent Laid-Open H9-3132 bulletin
Patent Document 5: Japanese Patent Laid-Open H10-298240 bulletin
Patent Document 6: Japanese Patent Laid-Open H5-222064 bulletin
Patent Document 7: Japanese Patent Laid-Open S63-295593 bulletin
Non-Patent Document 1: Jian R. et al. Langmuir 2001, 17, 3382-3389